Revertants of an S49 cell mutant that expresses altered cyclic AMP-dependent protein kinase

1982 ◽  
Vol 2 (10) ◽  
pp. 1229-1237
Author(s):  
T van Daalen Wetters ◽  
P Coffino
Keyword(s):  
Protein Kinase ◽  
Regulatory Subunit ◽  
Mutational Event ◽  
Wild Type ◽  
Cell Mutant ◽  
Dependent Protein Kinase ◽  
Cell Extracts ◽  
Counter Selection ◽  
Dependent Protein ◽  
Gene Lesion

Dibutyryl adenosine 3',5'-phosphate (Bt2cAMP)-sensitive (Bt2cAMPS) revertants were isolated from a resistant S49 cell mutant carrying a structural gene lesion in the regulatory subunit of cAMP-dependent protein kinase (cA-PK). This was accomplished with a counter-selection in which, first, Bt2cAMP was used to reversibly arrest revertants, and then a sequence of treatments with bromodeoxyuridine, 33258 Hoechst dye, and white light was used to kill cycling mutant cells. Reversion rates in nonmutagenized cultures could not be accurately measured, but spontaneous revertants do occur and with frequencies of less than 10(-7) to 10(-5). The mutagens ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitro-soguanidine (MNNG), and ICR191 increased the reversion frequency. In all cases, reversion to Bt2cAMP sensitivity was associated with restoration of wild-type levels and apparent activation constant for cAMP of cA-PK. MNNG induced revertants whose cell extracts contained cA-PK activity distinguishable from that of wild type by thermal liability. EMS did not. The counter-selection effectively isolates rare phenotypes and is therefore a useful tool in further somatic genetic experiments. The association of reversion with alterations in cA-PK function supports all previous data from this and other laboratories implicating cA-PK as the intracellular mediator of cAMP effects. Reversion is probably the result of a mutational event. Induction of reversion by ICR191 suggests the existence of a novel mechanism for generating revertants in somatic cells.

scholarly journals Revertants of an S49 cell mutant that expresses altered cyclic AMP-dependent protein kinase.

1982 ◽  
Vol 2 (10) ◽  
pp. 1229-1237 ◽  
Author(s):  
T van Daalen Wetters ◽  
P Coffino
Keyword(s):  
Protein Kinase ◽  
Regulatory Subunit ◽  
Mutational Event ◽  
Wild Type ◽  
Cell Mutant ◽  
Dependent Protein Kinase ◽  
Cell Extracts ◽  
Counter Selection ◽  
Dependent Protein ◽  
Gene Lesion

Dibutyryl adenosine 3',5'-phosphate (Bt2cAMP)-sensitive (Bt2cAMPS) revertants were isolated from a resistant S49 cell mutant carrying a structural gene lesion in the regulatory subunit of cAMP-dependent protein kinase (cA-PK). This was accomplished with a counter-selection in which, first, Bt2cAMP was used to reversibly arrest revertants, and then a sequence of treatments with bromodeoxyuridine, 33258 Hoechst dye, and white light was used to kill cycling mutant cells. Reversion rates in nonmutagenized cultures could not be accurately measured, but spontaneous revertants do occur and with frequencies of less than 10(-7) to 10(-5). The mutagens ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitro-soguanidine (MNNG), and ICR191 increased the reversion frequency. In all cases, reversion to Bt2cAMP sensitivity was associated with restoration of wild-type levels and apparent activation constant for cAMP of cA-PK. MNNG induced revertants whose cell extracts contained cA-PK activity distinguishable from that of wild type by thermal liability. EMS did not. The counter-selection effectively isolates rare phenotypes and is therefore a useful tool in further somatic genetic experiments. The association of reversion with alterations in cA-PK function supports all previous data from this and other laboratories implicating cA-PK as the intracellular mediator of cAMP effects. Reversion is probably the result of a mutational event. Induction of reversion by ICR191 suggests the existence of a novel mechanism for generating revertants in somatic cells.

Reversion of an S49 cell cyclic AMP-dependent protein kinase structural gene mutant occurs primarily by functional elimination of mutant gene expression

1983 ◽  
Vol 3 (2) ◽  
pp. 250-256
Author(s):  
T van Daalen Wetters ◽  
P Coffino
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Amino Acid ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Dibutyryl Camp ◽  
Dependent Protein Kinase ◽  
Type Function ◽  
Dependent Protein

The regulatory subunits of cyclic AMP (cAMP)-dependent protein kinase from a dibutyryl cAMP-resistant S49 mouse lymphoma cell mutant, clone U200/65.1, and its revertants were visualized by two-dimensional polyacrylamide gel electrophoresis. Clone U200/65.1 co-expressed electrophoretically distinguishable mutant and wild-type subunits (Steinberg et al., Cell 10:381-391, 1977). In all 48 clones examined, reversion of the mutant to dibutyryl cAMP sensitivity was accompanied by alterations in regulatory subunit labeling patterns. Some spontaneous (3 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (2 of 11) revertants retained mutant subunits, but these were altered in charge, degree of phosphorylation, or both. The charge alterations were consistent with single amino acid substitutions, suggesting that reversion was the result of second-site mutations in the mutant regulatory subunit allele that restored wild-type function, although not wild-type structure, to the gene product. The majority of spontaneous (8 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (9 of 11) revertants and all of the revertants induced by ethyl methane sulfonate (14 of 14) and ICR191 (12 of 12) displayed only wild-type subunits. Dibutyryl cAMP-resistant mutants isolated from several of these revertants displayed new mutant but not wild-type subunits, suggesting that the revertant parent expresses only a single, functional regulatory subunit allele. The mutant regulatory subunit allele can, therefore, be modified in two general ways to produce revertant phenotypes: (i) by mutations that restore its wild-type function, and (ii) by mutations that eliminate its function.

scholarly journals Deletion of the Regulatory Subunit of Protein Kinase A in Aspergillus fumigatus Alters Morphology, Sensitivity to Oxidative Damage, andVirulence

2006 ◽  
Vol 74 (8) ◽  
pp. 4865-4874 ◽  
Author(s):  
Wei Zhao ◽  
John C. Panepinto ◽  
Jarrod R. Fortwendel ◽  
Lauren Fox ◽  
Brian G. Oliver ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Protein Kinase ◽  
Oxidative Damage ◽  
Aspergillus Fumigatus ◽  
Fungal Pathogens ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Opportunistic Fungal Pathogen

ABSTRACT Aspergillus fumigatus is an important opportunistic fungal pathogen. The cAMP-dependent protein kinase (PKA) signaling pathway plays an important role in regulating morphology, growth, and virulence in a number of fungal pathogens of plants and animals. We have constructed a mutant of A. fumigatus that lacks the regulatory subunit of PKA, pkaR, and analyzed the growth and development, sensitivity to oxidative damage, and virulence of the mutant, along with those of the wild type and a complemented mutant. Both growth and germination rates of the mutant are reduced, and there are morphological abnormalities in conidiophores, leading to reduced conidiation. Conidia from the ΔpkaR mutant are more sensitive to killing by hydrogen peroxide, menadione, paraquat, and diamide. However, the hyphae of the mutant are killed to a greater extent only by paraquat and diamide, whereas they are less susceptible to the effects of hydrogen peroxide. In an immunosuppressed mouse model, intranasally administered conidia of the mutant are significantly less virulent than those of the wild type or a complemented mutant. Unregulated PKA signaling is detrimental to the virulence of A. fumigatus, perhaps through the reduced susceptibility of the mutant to damage by oxidizing agents and reduced growth kinetics.

scholarly journals Reversion of an S49 cell cyclic AMP-dependent protein kinase structural gene mutant occurs primarily by functional elimination of mutant gene expression.

1983 ◽  
Vol 3 (2) ◽  
pp. 250-256 ◽  
Author(s):  
T van Daalen Wetters ◽  
P Coffino
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Amino Acid ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Dibutyryl Camp ◽  
Dependent Protein Kinase ◽  
Type Function ◽  
Dependent Protein

The regulatory subunits of cyclic AMP (cAMP)-dependent protein kinase from a dibutyryl cAMP-resistant S49 mouse lymphoma cell mutant, clone U200/65.1, and its revertants were visualized by two-dimensional polyacrylamide gel electrophoresis. Clone U200/65.1 co-expressed electrophoretically distinguishable mutant and wild-type subunits (Steinberg et al., Cell 10:381-391, 1977). In all 48 clones examined, reversion of the mutant to dibutyryl cAMP sensitivity was accompanied by alterations in regulatory subunit labeling patterns. Some spontaneous (3 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (2 of 11) revertants retained mutant subunits, but these were altered in charge, degree of phosphorylation, or both. The charge alterations were consistent with single amino acid substitutions, suggesting that reversion was the result of second-site mutations in the mutant regulatory subunit allele that restored wild-type function, although not wild-type structure, to the gene product. The majority of spontaneous (8 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (9 of 11) revertants and all of the revertants induced by ethyl methane sulfonate (14 of 14) and ICR191 (12 of 12) displayed only wild-type subunits. Dibutyryl cAMP-resistant mutants isolated from several of these revertants displayed new mutant but not wild-type subunits, suggesting that the revertant parent expresses only a single, functional regulatory subunit allele. The mutant regulatory subunit allele can, therefore, be modified in two general ways to produce revertant phenotypes: (i) by mutations that restore its wild-type function, and (ii) by mutations that eliminate its function.

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